Development of metallothionein-based assays for the measurement of zinc status in humans

107 Pages·1997·3.7 MB·English

by  Sullivan, Vicki K.

DEVELOPMENTOFMETALLOTHIONEIN-BASEDASSAYSFORASSESSMENT
OFZINCSTATUSINHUMANS
By
VICKIK.SULLIVAN
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
1997
Copyright1997
by
VickiK.Sullivan
Tomyparentsandmyhusband.
ACKNOWLEDGMENTS
IthankmymentorDr.RobertCousinsforallowingmetolearnandaccomplish
exactlywhatIhadhopedtowhenIarrivedherefouryearsago,forexposingmetoa
laboratorythatoffersavastarrayoftechniquesinbasicandappliedscience,andfor
providinganunlimitedamountofhistime. Ialsothankmycommitteemembers,Drs.Gail
Kauwell,SusanS.Percival,RachelShiremanandKuo-JangKaofortheirexcellent
expertiseandguidancethroughoutthisexperience. IalsothankDr.Cousinsfor
surroundingmewithagroupoftalentedandcompetentco-workers. Ithankthefollowing
peoplefortheirsupport: BarbaraDavis,ChristinaKhoo,LorraineLangerhan-Foster,
SteveDavis,andKarolSmithforsharingthisexperiencewithme;MelissaTuasonforher
helpwiththehumanstudy;Drs.RayBlanchard,NoraHalloquistandRobertMacMahon
fortheirencouragementandexpertise;WarrenClarkforputtingupwithsharingalab
benchwithme,andVirginiaMauldinforalwaysgoingtheextramile.
Ithankbothofmyparentsfortheirunconditionallove,andforteachingmeby
examplethevalueofhardwork,persistence,andhonesty. Ialsothankthemforalways
listening. IthankmyhusbandShawnforhisloveanddevotion,foragreeingtoridethis
roller-coaster(graduateschool)withmeforsomanyyears,andforstayingontheride
throughalltheupsanddowns. IalsothankmyfurryfriendsSpot,ChewyandPumpkin,
formakingmelaughwhennooneelsecould,andforremindingmethatthesimplethings
inlifearetrulythemostimportant.
IV
TABLEOFCONTENTS
ACKNOWLEDGEMENTS iv
LISTOFFIGURES vii
ABBREVIATIONS »
ABSTRACT x
CHAPTERS
1 INTRODUCTION 1
2 REVIEWOFTHELITERATURE 5
Zinc 5
DistributionandFunctions 5
AbsorptionandTransport 6
Sources.BioavailabilityandRequirements 7
Deficiency 8
Toxicity 9
AssessmentofZincStatus 10
H
Metallothionein
MetallothioneinfortheMeasurementofZincStatus 13
ReverseTranscriptasePolymeraseChainReaction 13
3 MATERIALSANDMETHODS 17
RefinementofHumanMetallothioneinELISA 17
CompetitiveELISA 17
ELISAPlates 17
SourceofMetallothionein 17
PrimaryAntibody 19
ErythrocyteLysatePreparation 23
ProteinQuantitation 24
PlasmaZincandCopper 24
SandwichELISA 24
DevelopmentofCompetitiveReverseTranscriptasePolymeraseChain
ReactionAssay 26
CellCulture 25
PreliminaryHumanStudy 26
IsolationofMonocytes 26
ExtractionandPurificationofRNA 27
ReverseTranscriptasePolymeraseChainReactionAssays 28
CompetitiveRT-PCR 29
LongitudinalHumanStudy 35
ExperimentalDesign 36
AnalyticalProcedures 36
StatisticalAnalysis 37
4 RESULTS
RefinementofHumanMetallothioneinELISA 41
ELISAPlates 41
Antibodies 41
ErythrocyteLysatePreparation 42
SourceofMetallothionein 42
PreliminaryExperimentsforCompetitiveRT-PCRAssayandSandwich
ELISA 50
THP-1Cells 50
FlowCytometry 50
CompetitiveRT-PCR 50
SandwichELISA 54
LongitudinalHumanStudy 54
CompetitiveRT-PCR 54
SandwichELISA 55
PlasmaZinc 55
PlasmaCopper 56
5 DISCUSSIONANDCONCLUSIONS 62
APPENDIXA 73
APPENDIXB 80
REFERENCE 81
BIOGRAPHICALSKETCH 92
VI
LISTOFFIGURES
Figure gage.
3-1. Diagramofcompetitivereversetranscriptasepolymerasechainreaction(RT-
PCR)assay 31
3-2. Overviewofmetallothionein(MT)cDNAcompetitortemplateconstruction 32
3-3. Themetallothionein(MT)cDNAandcompetitorcDNAwereresolvedonan
8%polyacrylamidegelstainedwithethidiumbromide 33
3-4. Competitivereversetranscriptasepolymerasechainreaction(RT-PCR)assay
toquanifymetallothionein(MT)mRNA 34
3-5. Overviewofprotocolforlongitudinalhumanstudy 38
3-6. Competitivereversetranscriptasechainreaction(RT-PCR)assaytoquantify
metallothionein(MT)mRNA 39
3-7. PercentintegratedintensityforcDNAbandsaftercompetitiveRT-PCR 40
4-1. TitrationCurveforAnti-HumanMetallothioneinChickenAntibodies 43
4-2. SandwichELISAtoTestforHumanMetallothioneinSpecificity 44
4-3. SandwichELISAforComparisonofMonoclonalAntibodies 45
4-4. DirectELISAfortheComparisonofMTFromDifferentSpecies 46
4-5. EffectofProteaseInhibitorsonErythrocyteLysateMTStabilityOverTime 47
4-6. Zinc-stimulatedincreaseinmetallothioneinmRNAlevelsinTHP-1cellsusing
semi-quantitativereversetranscriptasepolymerasereaction(RT-PCR) 48
4-7. Flowcytometryprofileofhumanmonocytes 49
4-8. HumanMonocyteMTcDNAafterCompetitiveRT-PCR 51
4-9. SandwichELISAforErythrocyteMetallothionein 52
vn
4-10. StandardCurveforSandwichELISA 53
4-11. CompetitiveRT-PCR: MonocyteMetallothioneincDNA 57
4-12. Polyacrylamidegelelectrophoresisshowingintra-assayvariationfor
competitiveRT-PCRassay 58
4-13. Erythrocytehumanmetallothionein(hMT)asanalyzedbysandwichELISA 59
4-14. PlasmaZinc 60
4-15. CopperZinc 61
5-1. ComparisonofMonocyteMTmRNAandErythrocyteMTproteinResponse
toZincSupplementation 70
Vlll
ABBREVIATIONS
bp basepair
BSA bovineserumalbumin
cDNA complementarydeoxyribonucleicacid
ddH2 glassdistilleddeionizedwater
DEPC diethylpyrocarbonate
DMEM Dulbecco'smodificationofEagle'smedium
DNA deoxyribonucleicacid
dNTP deoxynucleosidetriphosphate
ELISA enzyme-linkedimmunosorbentassay
FAA flameatomicabsorption
FACS fluorescenceactivatedcellsorter
FBS fetalbovineserum
FITC fluorosceinisothyocyanate
FPLC fastproteinliquidchromatography
IACUC InstitutionalAnimalCareandUseCommittee
ICBR InterdisciplinaryCenterforBiotechnologyResearch
IgG immunoglobulinG
hMT humanmetallothionein
mRNA messengerribonucleicacid
MT metallothionein
NIH NationalInstituteofHealth
PAGE polyacrylamidegelelectrophoresis
PBS phosphate-bufferedsaline
PCR polymerasechainreaction
RIAs radioimmunoassays
RT-PCR reversetranscriptasepolymerasechainreaction
IX
AbstractofDissertationPresentedtotheGraduateSchool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
DEVELOPMENTOFMETALLOTfflONEINBASEDASSAYSFORTHE
MEASUREMENTOFZINCSTATUSINHUMANS
By
VickiK.Sullivan
May,1997
Chairman: RobertJ.Cousins
MajorDepartment: FoodScienceandHumanNutrition
Whilezincisanessentialmineralwithnumerousphysiologicalroles,areliable
indexforzincstatusinhumanshasnotbeendeveloped. Metallothionein(MT),acysteine-
richproteinoflowmolecularweight,isinducedbyzinc. BecausetheMTgeneis
transcriptionallyregulatedbyzinc,MThasgreatpotentialforservingasanindexof
dietaryzincstatus. Thespecificaimsofthisprojectwereasfollows: 1)todevelopanew
enzyme-linkedimmunoassay(ELISA)forthemeasurementofhumanMT(hMT)in
erythrocytes;2)todevelopamethodformeasuringhMTmRNAlevelsinmonocytes;and
3)totestthesemethodsinahumanzincsupplementationstudy. AsandwichELISAwas
developedforthemeasurementofhMTinerythrocytes. Acompetitivereverse
transcriptasepolymerasechainreaction(RT-PCR)assaywasdevelopedforthe
measurementofmonocytehMTmRNA. Twenty-fivehealthymalesubjectsparticipatedin
an18dayzincsupplementationstudy. ErythrocytehMT,monocytehMTmRNA,plasma
zincandplasmacopperwereevaluated. Zincsupplementationresultedinasignificant